Etiology

Purification and Properties of Foxtail Mosaic Virus

A. Q. Paulsen and C. L. Niblett

Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. Done in part by the senior authorat the Department of Plant Pathology, University of The Philippines at Los Baflos, Laguna, The Philippines.

We gratefully acknowledge gifts of antisera and viruses by W. Langenberg, D. Purcifull, and J. Shepard.Contribution No. 658, Department of Plant Pathology, Kansas Agricultural Experiment Station.Accepted for publication 23 March 1977.

ABSTRACT

PAULSEN, A. Q., and C. L. NIBLETT. 1977. Purification and properties of foxtail mosaic virus. Phytopathology 67:1346-1351.

Foxtail mosaic virus (FMV) infected 56 grass and 35 di- extraction and resuspension reduced aggregation. Purifiedcotyledonous species. Development of systemic symptoms FMV had an A 2 6 0/ 2 80 ratio of 1.2 and was very stable at 4 C.first on maturing leaves rather than on emerging leaves was Two components sedimenting at 122S and 144S wereunique and characteristic. Foxtail mosaic virus was detected. Extracted nucleic acid sedimented at 32S and itsseedborne in Briza maxima (2%) and Clintland oats (1%). infectivity was eliminated by RNase treatment. Leaf-dip andMyzus persicae and Nephotettix impicticeps were not purified preparations contained rod-shaped particlesvectors. Crude sap dilution endpoint was 10-6, thermal averaging 430 X 15 nm. Serological tests showed noinactivitation point 70 C, longevity in vitro was 46 days in relationship between FMV and barley stripe (BSMV), bromeundiluted sap and 105 days in buffered extract. Foxtail mosaic (BMV), clover yellow mosaic, panicum mosaic,mosaic virus was purified from Reno barley by chloroform- papaya mosaic, potato X, or tobacco mosaic viruses. Cross-butanol clarification and differential centrifugation. Igepon- protection tests showed no relationship between FMV andT-73 and sucrose added to the phosphate buffer for BSMV or BMV.

Additional key words: foxtail, Setaria viridis, S. italica, grass viruses.

A sap-transmissible virus was isolated in 1967 from the daylength during winter.foxtails Setaria viridis (L.) Beauv. and S. italica (L.) Seed transmission.--Several species that wereBeauv. with mildly chlorotic striped leaves. The plants systemically infected in the host range trials were allowedwere collected in a cornfield in Leavenworth, Kansas. to set seed in the greenhouse. Approximately 1,000Assays on indicator hosts for several sap-transmissible seedlings of each species were grown and tested for virusgrass viruses suggested that this virus differed from by inoculation to C. amaranticolor.known grass viruses. We designated this virus foxtail Virus purification.--Foxtail mosaic virus wasmosaic virus (FMV). Based on earlier data (7, 8) and that inoculated to 10- to 12-day-old Reno barley seedlings andreported here, we concluded that FMV is distinct from both inoculated and systemically-infected leaves wereother known sap-transmissible grass viruses, harvested 21-30 days later. Leaves were cut into short

strips and ground in small batches in a Waring BlendorMATERIALS AND METHODS with 1.5 ml of cold 0.03 M phosphate buffer, pH 8.0, and

1% 2-mercaptoethanol/g tissue. The liquid was squeezedFoxtail mosaic virus was maintained on either Pawnee through cheesecloth and used to grind additional batches.

wheat (Triticum aestivum L.) or Reno barley (Hordeum An equal volume of chloroform:butanol (1:1, v/v)vulgare L.). Inoculum routinely was prepared from plants mixture was added slowly with gentle stirring. Theinfected 1-4 mo. One part leaves was ground with mortar emulsion was kept at 4 C for 30-60 min then centrifugedand pestle in 15-20 parts 0.03 M potassium phosphate 10 min at 5,000g. The aqueous phase was filtered throughbuffer, pH 8.0, containing 2% Celite and 1% 2-mercapto- glass wool and centrifuged 1.5 hr at 147,000 g. Pelletsethanol. The extract was filtered through cheesecloth and were resuspended in 3-4 ml 0.02 M phosphate buffer, pHrubbed on Carborundum-dusted leaves with a 7.5, overnight at 4 C, and resuspension was completed thecheesecloth pad. In virus characterization experiments, next day by gently stirring with a glass rod. Theinoculum was prepared only from leaves with distinct preparations were pooled and centrifuged 10 min atmosaic symptoms and dilutions were made with 0.03 M 12,000 g. After two additional cycles, of differentialphosphate buffer, pH 8.0. All plants were grown from centrifugation (60 min at 269,000 g, then 10min at 12,000seed and inoculated while very young. Back-inoculations g), final pellets were resuspended in buffer (2-3 ml/ 100 gand other bioassays were made on half-leaves of tissue). These were combined, centrifuged 10 min atChenopodium amaranticolor Coste & Reyn. All 12,000 g, and the resulting supernatant liquid (purifiedinoculations were done above 24 C. Plants were grown virus) was stored at 4 C.under supplemental light that provided a 12- to 14-hr An alternative procedure involved grinding tissue in 0.5

M phosphate buffer, pH 7.5, containing 0.1% Igepon T-Copyright © 1977 The American Phytopathological Society, 3340 73, 1% 2-mercaptoethanol and 1% sucrose. Antifoam APilot Knob Road, St. Paul, MN 55121. All rights reserved. (Dow Corning) was sprayed into the Blendor jar before

1346

November 1977] PAULSEN AND NIBLETT: FMV CHARACTERIZATION 1347

TABLE 1. Symptoms induced by foxtail mosaic virus on mechanically inoculated hosts

Family and species Symptomsa

AmaranthaceaeCelosia plumosa Hort. CLGomphrena globosa L. RL

ApocynaceaeVinca rosea L. ISL

ChenopodiaceaeBeta vulgaris L. RLChenopodium album L. CL, NL, SCR, LPC. amaranticolor Coste & Reyn. CL, NL, SCR, LPC. ambrosioides L. RL, SCR, LPC. capitatum (L.) Asch. SL, NL, SCR, LPC. foetidum Lam. CL, NL, SMC. hybridum L. CL, NL, SM, LPC. murale L. NL, SMC. quinoa Willd. CL, NL, SMC. rubrum L. CL, NL, SCR, LPC. urbicum L. CL, CLR, SCR, LPSpinacea oleracea L. CL, NL, CLR, ISS

CompositaeZinnia elegans Jacq. ISL

CruciferaeArabis alpina L. ISLBrassica juncea (L.) Coss ISL

CucurbitaceaeCucumis sativus L. ISL

GramineaeAegilops bicornis Jaub & Spach. CL, SMA. cylindrica Host. CL, SMA. juvenalis L. CL, SMA. triaristata Ref. E Bertol. CL, SMA. triuncialis L. CL, SMAgropyron elongatum (Host) Beauv. ISLAlopercurus pratensis L. ISLAndropogon caucasius Trin. ISLA. halli Hack ISLA. ischaemum L. ISLAvena sativa L. SMA. sterilis L. SMBouteloua curtipendula (Michx.) Torr. SMB. gracilis (HBK) Lag. & Steud. ISSBriza maxima L. SMBromus inermis Leyss ISLBuchl6e dactyloids (Nutt.) Engelm. SMCoix lacryma-jobi L. ISSCynodon dactylon (L.) Pers. SMDigitaria sanguinalis (L.) Scop. ISSEchinochloa crus-galli (L.) Beauc. ISSHordeum vulgare L. SMLagurus ovatus L. SMOryza sativa L. ISSPanicum anceps Michx. SMP. antidotale Retz. SMP. capillare L. SMP. decompositum L. SMP. maximum Jacq. SMP. miliaceum L. SMP. ramosum L. SMP. scribnerianum Nash SMP. stapfianum Fourc. SMP. turgidum Forsck. SMP. virgatum L. SM

1348 PHYTOPATHOLOGY [Vol. 67

TABLE 1. (continued)

Family and species Symptomsa

Paspalum notatum Flugge ISLPennisetum glaucum (R.) Br. SMPhalaris arundinacea L. CLPhleum pratense L. ISLPoa pratensis L. ISLSecale cereale L. SMSetaria faberii Herm. SMS. italica (L.) Beauv. SMS. sphacelata (Schramm) Stapg. & Hubb. SMS. verticillata (L.) Beauv. SMS. viridis (L.) Beauv. SMSorghastrum nutans (L.) Nash SMSorghum bicolor (L.) Moench SMS. vulgare var. sudanense Hitch. SMTripsacum dactyloides L. SMTriticum aestivum L. CL, SMT. dicoccum Schrank CL, SMT. durum Desf. CL, SMT. monococcum L. CL, SMT. timopheevi Zhukolv. CL, SMZea mays L. SM

LeguminosaeCassiaflorida Vahl. NLCrotalaria spectabilis Roth. NLCyamopsis tetragonoloba (L.) Taub. NLGlycine max (L.) Merr. ISLV. sinensis (Torn.) Savi ISL

ScrophulariaceaeToreniafournieri Lindl. ISS

SolanaceaeLycopersicon esculentum Mill. ISSNicandra physaloides Gaertn. ISLNicotiana glauca Graham ISS, SCRN. megalosiphon Heurck & Muell. NL, ISS, SCRN. multivalvis Lindl. ISLN. raimondi L. ISSN. rustica L. NLN. tabacum L. ISSPhysalisfloridana Rydb. ISL

aAbbreviations for symptoms: CL, RL, or NL - chlorotic, red, or necrotic local lesions; CLR - chlorotic local rings; ISL or ISS -

infection symptomless - local (L) or systemic (S); LP - line patterns; SCR - systemic chorotic rings; and SM - systemic mosaic.

use to prevent foaming. Clarification and further 1) was mixed with nine parts of 1% sodium phos-purification were as described above and the final pellets photungstate (NaPT) and the mixture incubated 20-30were resuspended in 0.02 M phosphate, pH 7.5, min at 4 C. A drop of the mixture was placed on carboncontaining 0.1% Igepon T-73 and 1% sucrose. stabilized, Formvar-coated grids and viewed in a RCA-

Sedimentation coefficients of foxtail mosaic virus and EMU4 or Hitachi HU- lB electron microscope. Leaf-dip

foxtail mosaic virus nucleic acid.--Sedimentation preparations were made by touching a freshly cut, healthyor infected barley leaf to a drop of NaPT on a grid and

coefficients were determined using linear-log sucrose viewed similarly.gradients as described by Brakke and Van Pelt (2). Virus Serology.-One ml of purified FMV (A260 = 5.0 wasand RNA standards were prepared, analyzed, and emulsified with an equal volume of Difco Freund'sfractionated as previously described (6). All fractions incomplete adjuvant and injected intramuscularly into awere dialyzed overnight in 0.02 M potassium phosphate rabbit. Seven injections were given at 4-day intervals, andbuffer, pH 7.0. One-half of each dialyzed nucleic acid blood was sampled for antiserum at weekly intervals.fraction was incubated with 2 jIg ribonuclease A (RASE, Antiserum titer was determined by the microprecipitinWorthington Biochemical Corp., Freehold, NJ 07728) method (1) with FMV at A280 = 0.03. Routine assays forfor 30 min at 37 C. All preparations were inoculated to C. FMV and tests for relationships to some viruses wereamaranticolor with autoclaved cotton swabs. performed using the Ouchterlony double - diffusion

Electron microscopy.-One part purified FMV(A 260 = procedure (0.75% lonagar No. 2 in 0.05 M Tris-HC1, pH

November 1977] PAULSEN AND NIBLETT: FMV CHARACTERIZATION 1349

7.5, 0.85% NaC1, .02% NaN 3)(1). Relationships with seedlings. For persistent transmission trials, four to fivepotex viruses [potato virus X (PVX) clover yellow mosaic instar nymphs were permitted to feed on infected I R-20virus (CYMV) and papaya mosaic virus (PaMV)] were rice for 2 to 5 days. One to two leafhoppers wereinvestigated by three procedures; (i) agar double diffusion transferred to each test seedling using the test tube feedingfor both intact and pyrrolidine-disrupted virus (9), (ii) technique (5). Daily serial transfers were made to healthytube precipitin (1), and (iii) serologically specific electron seedlings for 10 days.microscopy (3).

Insect transmission.--Apterous Myzuspersicae (Sulz.) RESULTSadults were used to determine whether FMV might betransmitted in a stylet-borne or circulative manner to Host range, symptoms, and seedPawnee wheat and C. quinoa. For stylet-borne transmission.-Foxtail mosaic virus infected a wide hosttransmission trials the aphids were starved 30 min prior to range among the grasses and dicotyledonous speciesa 5 min access feeding period on FMV-infected wheat (Table 1). Of 68 grasses tested, only 12 were foundleaves. Ten aphids observed to probe within this time immune in repeated trials. They were Agropyron smithiiwere transferred to each test seedling and removed 3 hr Rydb., Andropogon halli Hack., A. intermedius Brown,later. For persistent transmission trials, five to ten adults Arrhenatherum elatius (L.) Mert. and Koch, Digitariafrom an established colony on infected C. quinoa or C. horizontalis Willd., Lolium perenne L., Phalarisamaranticolor were transferred to each young healthy C. arundinacea L., Sisyrinchium bellum Watts., Sorghumquinoa plant and removed after 24, 48, or 72 hr. halepense (L.) Pers., Sporobolus cryptandrus Gray, Stipa

The rice green leafhopper, NephotettixirnpicticepsIsh. viridula Trin., and Zoysia japonica Steud. Two otherwas used with the rice cultivars Taichung Native 1 and IR- monocots, a Commelina sp. and Cyperus esculentus L.,20 as assay and production hosts. For nonpersistent also were immune.transmission trials, male and female adults were starved Reactions of the 56 susceptible grasses ranged fromfor 2 hr then fed on infected rice leaves. After 30, 60, 120, localized to systemic infections, with many asand 180 min, two to five leafhoppers were transferred to symptomless hosts (Table 1). On Setaria species and most

susceptible grasses, the first symptoms were observed onemerging leaves as a chlorotic striping that persisted untilthe leaves became senescent. Wheat, rye, barley, andsusceptible corn cultivars showed distinct light-green toyellow eyespots which became golden or ashy white incolor at temperatures above 24 C.

Of 111 dicotyledonous species tested only 35 werefound to be susceptible. Susceptible dicotyledonousspecies either were symptomless hosts or only becameinfected locally (Table 1). Lesions frequently becamenecrotic at temperatures below 22 C. Except for Betavulgaris on which only local lesions were induced, allother species of Chenopodiaceae tested reacted with localand systemic infection. Only two other species, Nicotianaglauca and N. megalosiphon became systemicallyinfected with visible symptoms. On these hosts, systemicsymptoms as chlorotic rings and line patterns appeared10-14 days after inoculation. The sequence of appearanceof systemic symptoms on wheat, barley, rye, and relatedspecies, as well as on the Chenopodium spp. wascharacteristic. Local lesions as chlorotic spots developedon inoculated leaves in 5-6 days. Systemic symptomsappeared initially on the developed uninoculated leavesabove the inoculated ones, and much later on emergingleaves as they expanded and matured. The symptomlessemerging leaves, however, provided very infectiousextracts.

Best symptoms were observed at temperatures above24 C. At lower temperatures, local lesions frequentlybecame slightly necrotic, and symptom appearancegenerally was delayed in all hosts.

Based on over 1,000 seedlings grown per species andassayed on C. amaranticolor, no seed transmission wasdetected in C. amaranticolor, C. quinoa, Setaria italica,S. lutesecens, S. viridis, Panicum capillare, Reno barley,or Pawnee wheat. About 2% transmission was observed

Fig. 1. Electron micrograph (X 55,000) of foxtail mosaic virus in Briza maxima and 1% in Clintland oats (Avena sativa).particles purified from Reno barley. Bean pod mottle virus was On B. maxima some seedlings showed mosaic symptomsadded as an internal standard and has a diameter of about 30 nm. due to seed-transmitted FMV, but most did not. No

1350 PHYTOPATHOLOGY [Vol. 67

symptoms were observed on oat seedlings, but FMV (PMV), PaMV, PVX, and tobacco mosiac (TMV) virusesinfection was demonstrated by back inoculation to C. were reacted with purified FMV and crude extracts ofamaranticolor. plants infected with the different viruses. In addition,

Physical properties. -Extracts prepared from infected crude extracts of plants infected with FMV, CYMV,Pawnee wheat or Reno barley were diluted in 0.03 M PaMV, and PVX were mixed with an equal volume of 5%phosphate buffer, pH 8.0. A few local lesions were pyrrolidine and reacted similarly with antisera producedinduced at 10-6, but none at 10-7 dilution. Foxtail mosaic to protein subunits of CYMV, PaMV, and PVX.virus tolerated heating at 68 C but not 70 C for 10 min. Precipitin bands formed only between homologousUndiluted sap kept at 24-26 C was still infectious after 105 antigen: antibody combinations, except in the case of thebut not after 119 days. Some infectivity was lost when two PVX antisera, which reacted to both intact anddiced leaves were desiccated over CaC12. These samples disrupted PVX. In tube precipitin tests 0.5 ml of a 1:4were infectious only to 10-5 dilution after desiccation for dilution of all antisera was reacted with an equal volume21 days at 4 C, but contained infectious virus after 5 yr, of purified FMV or PVX (both I mg/ ml) or 0.5 ml of anthe longest time tested. Lyophilized and liquid-nitrogen extract from healthy or FMV-infected barley at 37 C for 2frozen tissues prepared by the American Type Culture hr and then stored overnight at 4 C. The barley tissue wasCollection (ATCC virus number PV-139) were also ground in 0.05 M Tris-HCi, pH 7.5, (5 g/ 10 ml), and theinfectious for at least 1 yr, extract was filtered through cheesecloth and centrifuged

Cross-protection tests.-Previous infection of C. (20 min, 30,000 g) before use. Abundant precipitate wasamaranticolor and Golden Cross Bantam sweet corn by observed in reactions between purified and crude FMVFMV did not protect such plants from subsequent and FMV antiserum and also between PVX and itsinfection by either brome mosaic (BMV) or barley stripe antiserum, but no other reactions were observed.mosaic (BSMV) viruses. Similarly, infection by BMV or Serologically specific electron microscopy was performedBSMV did not protect C. amaranticolor against FMV in all combinations using normal serum and antisera toinfection. CYMV, FMV, PaMV, and PVX and crude extracts as the

Purification and sedimentation coefficients.-Both source of each virus. Numerous virus particles werepurification techniques yielded opalescent preparations observed with all the homologous combinations. But withwith A260/280 = 1.2. The preparations were highly the heterologous combinations, few particles wereinfectious, producing 100 or more local lesions per half- observed and the number seldom exceeded that of theleaf at A260 = 0.03. Some precipitation was apparent after normal serum control.6 mo storage at 4 C, but preparations were still highly Insect transmission.-No evidence of FMVinfectious. transmission was obtained for either M. persicae or N.

None of several methods tried for purifying FMV impicticeps by either the stylet-borne or persistentresulted in total recovery of virus from the plant tissue. mechanism. In all experiments each test plant was backConsiderable virus (measured by infectivity) was lost at assayed to C. amaranticolor 3-4 wk after inoculation.each purification step and this was only partiallyprevented by adding Igepon T-73 and sucrose to the DISCUSSIONbuffer. Two opalescent and UV-absorbing componentswere detected after density-gradient centrifugation of Many new hosts of FMV and their reactions arepurified virus. These sedimented at 122S and 144S and reported. Hosts of FMV include both monocotyledonsboth were infectious. Analysis of nucleic acid and dicotyledons and its host range is extremely broad.preparations revealed a single, infectious, UV-absorbing However, FMV has not been isolated from crop plantscomponent sedimenting at 32S, and this component was and presently causes no known economic loss. Seedobtained from both the 122S and 144S components. transmission occurred in two hosts, but neither host isInfectivity of the nucleic acid preparations was eliminated widespread in Kansas. Therefore, FMV must persist fromby ribonuclease treatment. This suggests that FMV year to year in infected perennial hosts or be seed-contains one single-stranded RNA species with a transmitted in species we did not test. Neither the aphidmolecular weight of about 2.03 to 2.24 X 106 daltons (4, nor leafhopper tested could vector FMV. Therefore, the11). In comparative experiments FMV-RNA sedimented natural vector or method of dissemination of this virusslightly more rapidly than TMV-RNA which sediments at remains unknown.31S and has a molecular weight of 2.05 X 106 daltons (2, Foxtail mosaic virus is extremely stable in both crude11). and purified preparations and reaches high

Electron microscopy and serology. -Purified concentrations in several hosts. The extreme longevitypreparations of FMV contained particles that were rod- and dilution end point approach those reported for TMV,shaped and flexuous (Fig. 1). Several different lengths but the thermal inactivation temperature (70 C) is muchwere observed, but the most common particles measured lower than that of TMV (10).430 X 15 nm. Of over 1,000 particles that were measured, There is a marked tendency for FMV to form end-to-37% were this length. End-to-end aggregation of particles end aggregates and this increases with the degree ofwas very common in purified preparations. Leaf dip purity. Aggregation can be reduced somewhat bypreparations also contained many 430 X 15-nm particles, including Igepon and sucrose in the purification buffers.but aggregation was less frequent. Monomers or dimers generally were observed by electron

Antiserum prepared to purified FMV possessed a titer microscopy. This observation and the recovery of a singleof 1:1,280 when tested by the microprecipitin method 32S RNA species from both the 122S and 144S viruswith FMV at A28 0 = 0.03. In Ouchterlony agar diffusion components suggest that the 144S component is a dimertests antisera to BSMV, BMV, CYMV, panicum mosiac of the 122S component. With TMV and other viruses,

November 1977] PAULSEN AND NIBLETT: FMV CHARACTERIZATION 1351

aggregates of three, four, and often more particles are using serologically specific electron microscopy. Virologyobserved, but aggregates greater than dimers were not 56:652-653.seen in preparations of FMV. Therefore, loss of 4. HULL, R., M. W. REES, and M. N. SHORT. 1969. Studiesconsiderable virus at each purification step may result on alfalfa mosaic virus. I. The protein and nucleic acid.from interaction of FMV with host constituents, which Virology 37:404-415.are lateIr removed, rather than formation of large, readily 5. LING, K. C. 1966. Nonpersistence of the tungro virus of riceain its leafhopper vector, Nephotettix impicticeps.sedimented, aggregates of FMV. Phytopathology 56:1252-1256.

Our results indicate that FMV is distinct from other 6. NIBLETT, C. L., and A. Q. PAULSEN. 1975. Purificationknown sap-transmissible grass viruses, TMV, and the and further characterization of panicum mosaic virus.potex viruses. No serological relationship was Phytopathology 65:1157-1160.demonstrated between FMV and these viruses nor was 7. PAULSEN, A. Q. 1970. Reactions of eleven Chenopodiumthere cross protection between FMV and BSMV or species to seven sap-transmissible grass viruses.BMV. Other data such as host range, physical properties, Phytopathology 60:1307 (Abstr.).particle length, and sedimentation characteristics support 8. PAULSEN, A. Q., and W. H. SILL, JR. 1969. Hosts,

symptoms and characteristics of a sap-transmissible virusthe designation of foxtail mosaicvirus a's a distinct virus. isolated from foxtails. Phytopathology 59:1942-1943(Abstr.).

LITERATURE CITED 9. SHEPARD, J. F. 1972. Gel-diffusion methods for theserological detection of potato viruses X, S, and M.

1. BALL, E. M. 1961. Serological tests for the identification of Montana Agric. Exp. Stn. Bull. 662. Bozeman, Montana.plant viruses. Am. Phytopathol. Soc., St. Paul, Minn. 16 72 p.p. 10. SMITH, K. M. 1957. A textbook of plant virus diseases. 2nd

2. BRAKKE, M. K., and N. VAN PELT. 1970. Properties of ed. Churchill Ltd., London. 652 p.infectious ribonucleic acid from wheat streak mosaic 11. SPIRIN, A. S. 1963. Some problems concerning thevirus. Virology 42:699-706. macromolecular structure of ribonucleic acids. Prog.

3. DERRICK, K. S. 1973. Quantitative assay for plant viruses Nucleic Acid Res. 1:301-345.